The increasing popularity of single-use systems for bio-processing is apparent in the market place and can be readily understood by considering a typical biotech manufacturing facility. The infrastructure required to implement a facility using traditional glass/steel bioreactors, mixers, and purification systems is substantial, and the time and expense required to construct such bio-process systems can be immense. The requirement that both the equipment itself and also the ingress and egress tubing utilize inert materials such as 316L electro-polished stainless steel requires a large initial investment and the bioreactors, (i.e. bio-process vessels) mixers and down-stream processing equipment (e.g. chromatography skids, filtration systems) all have a considerable footprint. In contrast, the size and form factor of single-use platforms generally permits easier storage and re-configurability when compared to traditional, rigid glass/steel solutions. Other advantages of single use systems include a lower support infrastructure and time savings over traditional designs including specifically the reduction in preparation and sterilization time, the reduced need for purified water for cleaning the vessel after a run, and the significantly reduced post run maintenance time. Additionally, single use systems and their associated plastic tubing lend themselves to being re-configured and validated quickly and efficiently as manufacturing or process requirements change. In the context of the present invention we will focus primarily on single-use bioreactors, but the principals apply generically to any of the aforementioned single-use equipment used in bioprocessing, both in the upstream processing (USP) and downstream (DSP) processing arenas.
Although a number of different styles of single use bioreactors have been conceived and introduced into the marketplace, two types currently predominate. The first type of single-use bioreactor to become commercially popular is generally referred to as the “pillow” or “rocker” bag style, and is described, for example, in U.S. Pat. No. 6,190,913 the teaching of which is incorporated herein by this reference. This style of bag can be constructed from a variety of different polymeric materials, but generally speaking, low or ultra low density polyethylene (LDPE) and ethylene/vinyl-acetate (EVA) copolymers are currently among the most common materials for at least the innermost layer of the bag, i.e., the bag surface which is in contact with the aqueous growth medium. Other materials sometimes used in the construction of the single-use bioreactor vessels include high density polyethylene (HDPE) and Kevlar (Poly-paraphenylene terephthalamide). The pillow or rocker type of single-use bioreactor utilizes a wave motion induced by movement of a bag support platform which generally rocks about a single axis to both mix and sparge (aerate) the contents of the bioreactor. While the rocker type single-use bioreactor bag has enjoyed some marketplace success, to date one major issue has been the lack of robust, single-use sensors that can be integrated into these rocker bags and preferably be radiation sterilized together with the bag. By robust, we mean accurate, gamma or beta radiation stable and capable of being used for real time (real time within the speeds or time responses required for bio-processing) e.g. providing 1-3 second sampling for process monitoring and control for at least 21 days. The pillow or rocker bag is not the only type of single-use bioreactor vessel in use today. There are also single-use polymeric hard shell bioreactors that functionally imitate small scale glass vessels, and also larger scale single-use, plastic liner bags that fit inside both rigid pilot and production scale glass/stainless steel stirred tank bioreactors (see e.g., U.S. Pat. No. 7,384,783 the teaching of which is incorporated herein by this reference). The larger liner bags are typically constructed of multi-layer film laminates that also utilize ultra low density polyethylene or EVA for at least their innermost layer.
By way of example, FIG. 6 shows the construction of the CX-14 film used by Thermo Fisher Scientific for bioreactor vessels. Sensors are generally introduced into these larger single-use bioreactors through lateral ports. Both pillow (rocker) bags and liner bags can be considered to be “polymeric bioreactor vessels” for purposes of the utilization of the bioprocess monitoring assembly of the present invention.
One key issue affecting polymeric bioreactor vessels and other types of single-use equipment in both upstream and downstream applications in general has been the method by which to introduce sensors and ancillary monitoring equipment or assemblies that require multiple different materials (e.g.: mechanical assemblies). The sensors (both single-use and traditional multi-use) are often introduced through the type of prior art port shown in FIG. 1 (see e.g. published application US2006/0240546) or as is shown in FIG. 2 of this application specifically for use with a rocker bag. The port shown in FIG. 1 can be used to introduce into the vessel a monitoring assembly which allows the use of different types of sensing elements while the port shown in FIG. 2 is generally restricted to fiber optic based, single-use sensor systems. We use the term “port” herein to denote a part of our assembly to make it clear that we are replacing these prior art port and component assemblies compromised of these separate components. It should be noted, however, in the invention described here that there really is no longer a port or a separate monitoring component which enters into the vessel through said port. The concept of a port dates back to glass and stainless steel bioreactors where the ports were comprised of threaded openings in the headplate or the actual vessel wall and into which sensors or other components were secured by a threaded cap with an o-ring seal. A more accurate analogy here would be if the assemblies were welded into the stainless steel bioreactor wall or the steel headplate in order to effect a seal.
The prior art port shown in FIG. 1 is typically made of a material that is similar to the surface of the bag that it is in contact with, as this allows it to be readily fused (e.g., thermally or ultrasonically welded) to the bag surface. The port shown in FIG. 1 is comprised of a cylindrical tube 10 and a flange 11. This type of port uses a mechanical seal to prevent leakage around the normally cylindrical sensor or other object introduced into the tube portion of the port. This mechanical seal can be a friction fit created by surface to surface contact over a relatively large area as shown in FIG. 3 (see published application US 2006/0240546). FIG. 3 shows in more detail how a cylindrical object (e.g., a conventional 12 mm diameter electrochemical dissolved O2 (DO) or pH probe or aseptic connector like a KleenPak™) 34 fits into the tubular member 31 with a large contact area 33. This prior art port has a feature 32 that emulates an O-ring and an annular flange 35 which is welded to the liner of the single use bioreactor vessel. Similarly, the port can actually utilize an O ring seal as shown in FIG. 4, which shows a single use, free space optical assembly (e.g., single use sensor sheath) 41 installed in tubular port member 42 with a weldable polymer flange 43. The O-rings 44 are shown as residing in grooves 45 in the port tubular member. While these port designs can generally provide an air and water tight seal between the port and introduced assemblies, a significant amount of time is required to qualify and test these assemblies (e.g.: validate them for cGMP use) and they cannot be simply and directly assembled when manufacturing single-use polymeric bioreactor vessels. Additionally, there are circumstances where it is difficult to design a suitable port assembly to support the sensor assembly. This is especially true in the case of rocker type single-use bioreactor bags where there are drawbacks to using fiber optic based single-use sensors, but introducing an optimally designed free space based optical sensor assembly as described in U.S. Pat. No. 7,489,402, the teaching of which is incorporated herein by this reference, might require a larger port. However, a large port can put stress on the bag materials and is therefore difficult to construct such that the integrity of the bag can be assured while at the same time maintaining a leak free seal.
A preferable method of introducing sensor assemblies, (or other type of monitoring assembly) into single-use bioreactor vessels would be to simply weld these assemblies directly into the bags in a manner similar to the way that prior art ports and vents are presently attached to single-use bag liners. To date, this has not generally been feasible for most sensors or sensor assemblies. The reasons for the inability to implement such a solution for introducing sensors and other accessories into single-use bioreactor vessels or other single-use equipment include the fact that the bags or liners are generally made from laminated films where the inner layer is typically a high surface tension polymer such as ultra-low density LDPE or EVA; while the material used for the sensor assemblies (e.g.: whether a free space optical sensor, or electrical sensor) is generally needs to be a polymer such as a polycarbonate, cyclo-olefin, copolyester, or other thermo plastic that can be either transparent or opaque, substantially rigid, can meet USP Class VI standards, and in particular, can withstand the 50 kGy of gamma or beta radiation as is normally used for sterilization without a significant change in its physical or optical properties (e.g., the materials cannot become brittle or change opacity). The laminated films used to make the bags or vessel liners can be readily welded together, and although the prior art ports which are typically constructed from materials matching the inner layer of the single-use bioreactor (e.g.: LDPE, EVA, PVDF, or other polyolefin) can also be welded to the bag or liner, the optimal material for the sensor assembly itself cannot be readily welded to the film liner materials or to a port of the same material as the liner (See: “Materials of Construction for Single-Use Bioprocessing Systems”, William Hartzel, Innovations in Pharmaceutical Technology, p 46, April 2007). We have found that the two materials i.e. the sensor assembly and the bag liner cannot be melted or glued together without altering the surface of at least the contact layer of the liner and generally without altering both material surfaces. Therefore, the ability, as is enabled by the current invention, to construct complex assemblies that can be welded directly to the bag provides important new possibilities for putting sensors or assemblies in single-use bioreactor vessels and addresses the many issues present with existing sensor port designs. In addition, in order to fully enable the single-use paradigm and process optimization on a global scale, the automation software, hardware, and single-use sensors must be expanded from upstream processing (USP) units such as mixers and bioreactors to also encompass downstream bioprocessing (DSP) tools such as chromatography assemblies and filtration skids which utilize similar films. The advent of flexible, modular equipment with integrated data historization would allow the collection of a unified set of process data from buffer mixing to the final ultra-filtration of the bioprocess product. The availability of data from sensors of specific process modules (e.g., mixer, bioreactor, and different process configurations, especially on the downstream side), would allow users to develop models for each process step and the interactions therein. Once sufficient information becomes available from the database, the bio-process engineer could optimize the entire process end-to-end and implement yield modeling.
As used herein the term bioprocess vessel is intended to include both USP and DSP tools. In DSP the equipment would ideally implement single-use sensors fabricated using the manufacturing processes of the present invention as described herein to either replace traditional sensors and/or enable new additional analytical capability. DSP equipment that utilizes film technology similar to that utilized in USP polymeric bioprocess vessels is described in U.S. Pat. No. 7,935,253. Ideal “smart” sensors for the DSP as well as the USP would have the capability of being pre-calibrated and gamma or beta irradiation sterilized along with the bio-process vessel itself. In this way the sensors would arrive enclosed in the transport container together with the bio-process vessel. Thus, the entire system arrives sterile, thereby minimizing operator time during process setup. For example, in downstream applications, there is also a significant need for measuring pH and temperature, as well as optical density and product purity (e.g., viral load, biological impurities). In DSP sensor design, the ability to combine composite materials is particularly important as the optical requirements in the ultraviolet range further significantly limit the materials choices, and where requirements on extractables and leachables are becoming ever more stringent. Furthermore, as the size of single-use mixers, bioreactors, and DSP equipment increases to meet capacity, the risk of leakage from a standard sensor/port configuration increases with the greater pressure inside the container. This ability as provided by the present invention to utilize material combinations that were previously considered incompatible from a bonding perspective so as to provide an impermeable seal is an important enabling factor in both DSP and USP.